| Salmonella and Listeria moncytogenes are two of the most significant food-borne pathogenic bacteria worldwide which have a great risk to human health and food safety. Developing rapid detection methods of the two pathogenic bacteria are significant for controlling food contamination and food-borne diseases, and tracing the source after the outbreak, evaluating prevention and control measures and so on. Polymerase chain reaction (PCR)is one of the relatively mature fast detection techniques. There are few reports about foodborne pathogenic bacteria detection by enrichment-PCR. Real-time PCR (RT-PCR) is an emerging method in recent years, where adding fluorescent elements into the PCR system, could realize the PCR process monitoring and quantitative analysis on the initial template by the fluorescent signal accumulation, with sensitive specific, fast and efficient advantages. There had been some reports about detection of foodborne pathogenic bacteria by RT-PCR abroad, but there had been few reports in China.Specific primers and probes were chosed on the basis of virulence genes of Salmonella and Listeria moncytogenes. PCR and RT-PCR for detection of the two bacteria were developed and optimized by employing some conference strains. The sensitivity and specificity of enrichment-PCR and RT-PCR were evaluated through artificially contaminated experiments, and compared with that of tratidional method. The application of enrichment-PCR and RT-PCR were assessed by detecting some market-sold food samples. A duplicate RT-PCR for detection of Salmonella and Linsteria monocytogenes was established on the basis of single RT-PCR of the two bacteria.I Study on rapid detection techniques of salmonella in food1. Study on enrichment-PCR for detection of salmonella in foodSalmonella specific primers were chosed on the basis of invA gene. PCR method was developed and optimized by employing 48 conference strains.30 strains of salmonella tested were positive, and 18 strains of non-salmonella tested were negative, indicating the PCR established was stable and specific. Poultry samples were artificially contaminated with final inoculated concentration(1,10,102,103,104,105 and 106 CFU per 25g)of Salmonella CMCC50041. All samples were incubated for 0 h,4 h,8 h,12 h and 18 h and 1mL of culture solutions were taken from the samples to extract DNA, respectively. PCR and traditional methods were applied to detect Salmonella bacteria and their sensitivity and specificify were compared. PCR detection limit for artificially contaminated samples after enriching for 12h was 1 CFU/25g. Traditional method detection limit was 10 CFU/25g. The time limit of enrichment-PCR is four days shorter than that of traditional method at least. Sixteen samples of retail whole poultry collected from markets in Beijing were detected by the above two methods after enriching for 12 h in SC. The positive rate was 43.75% (7/16)by enrichment-PCR,which was same with that by traditional method. Enrichment-PCR is suitable for rapid and qualitative detection salmonella in poultry.2. Study on real-time PCR for detection of salmonella in foodA RT-PCR method for detection salmonella was developed with specific primers and Taqman probe on the basis of invA gene, and all 30 strains of Salmonella tested were positive, and all 18 non-Salmonella strains were negative, indicating the method was highly specific for the detection of Salmonella. Serial 10-fold diluted suspension culture of CMCC 50041 were detected by RT-PCR, and quantification was possible over a 8-log dynamic range(5.2×103CFU/ml?5.2×1010CFU/ml),with a limit of 5.2×103CFU/ml. Correlation coefficients of standard curves constructed using the cycle at threshold (Ct) versus log value of concentration of Salmonella showed good linearity (R2=0.999, e=0.99). Poultry samples were artificially contaminated with different concentration of Salmonella CMCC50041 and detected by RT-PCR after enrichment in SC for various time, and detected by PCR and traditional method at the same time. Detection limit and time limit of the three methods were compared. The results showed that before enrichment for 8h, the sensitivity of RT-PCR was 10-100 times more than that of PCR, and after enrichment in SC for 12h,RT-PCR and PCR could detect as few as 1CFU/25g, which is 10 times more than the sensitivity of traditional method. Standard curve of sample after enrichment for 12h was established, which is Conc= 10^(-0.305×Ct12h+ 10.870), and R212h=0.995, e12h=1.02. There was an excellent correspondence between the predicted and the given numbers of CFU per 25g sample (Var?20%). Sixteen poultry samples were analyzed for the presence of salmonella by RT-PCR, enriment-PCR and traditional methods. The results of the three methods are no differences, all of them tested the same 7 positive samples and the positive rate is 43.75%. The positive ones could be quantitative analyzed and determined the initial salmonella numbers of CFU/25g.The established RT-PCR technology is suitable to rapid detect salmonella in poultry samples, and the process can be finished in 15h.?. Study on rapid detection techniques of Listeria moncytogenes in food1. Study on enrichment-PCR for detection of Listeria moncytogenes in foodListeria moncytogenes specific primers were chosed on the basis of hlyA gene. PCR method was developed and optimized by employing 46 conference strains.26 strains of salmonella tested were positive, and 20 strains of non-salmonella tested were negative, indicating the PCR established was stable and specific. Pork samples were artificially contaminated with final inoculated concentration(1.3,13,1.3×102,1.3×103,1.3×104, 1.3 ×105 and 1.3×106CFU per 25g)of Listeria moncytogenes CMCC54004. All samples were enrichment for 0h,4 h,8 h,12 h,18 h,30h,36h and 46h, and lmL of culture solutions were taken from the samples to extract DNA, respectively. PCR and traditional methods were applied to detect Listeria moncytogenes and their sensitivity and specificify were compared. After enriching for 46h, the PCR detection limit for artificially contaminated samples was 1.3CFU/25g, which is the same with traditional method detection limit. The time limit of enrichment-PCR is four days shorter than that of traditional method at least. Twenty-four pork samples detected by the above two methods after enriching for 46 h, Both of them tested the same 17 positive samples and the positive rate is 70.83%.2. Study on real-time PCR for detection of Listeria moncytogenes in foodA real-time PCR method for detection Listeria moncytogenes was developed with specific primers and Taqman probe on the basis of hlyA gene, and all 26 strains of 1Listeria moncytogenes tested were positive, and all 20 non-Listeria moncytogenes strains were negative, indicating the method was highly specific. Serial 10-fold diluted suspension culture of CMCC 54004 were detected by RT-PCR, and quantification was possible over a 7-log dynamic range(1.3×l03CFU/ml?1.3×109CFU/ml), with a limit of 1.3×103CFU/ml.Correlation coefficients of standard curves constructed using Ct versus log value of concentration of Listeria moncytogenes showed good linearity (R2=0.999, e=0.97). Pork samples were artificially contaminated with different concentration of Listeria moncytogenes CMCC54004 and detected by RT-PCR after enrichment in LB for various time,and detected by traditional method and PCR at the same time. Detection limit and time limit of the three methods were compared. The results showed that all of them could detect as few as 1.3CFU/25g pork sample, and just single-step enrichment for 24h were required by RT-PCR, while two-steps enrichment for about 46h were needed by traditional method and PCR to reach the same detection limit. Standard curve of sample after enrichment for 24h was established, which is Conc= 10^(-0.298×Ct24h+l 1.606), and R224h=0.998, e24h=0.99. There was an excellent correspondence between the predicted and the given numbers of CFU per 25g sample (Var?15.58%). Twenty-four pork samples were analyzed for the presence of Listeria moncytogenes by real-time PCR, enrichment-PCR and traditional methods. The results of the three methods are no differences, all of them tested the same 17 positive samples and the positive rate is 70.83%. The positive ones could be quantitative analyzed and ascertained the initial Listeria moncytogenes numbers of CFU/25g.The established RT-PCR technology here is suitable to rapid detect Listeria moncytogenes in pork samples, and the process can be finished in 27h.? The real-time PCR method for simultaneous detection of Salmonella and Listeria monocytogenes were developedA real-time PCR assay was developed for detecting Salmonella and Listeria moncytogenes simultaneously on the basis of single RT-PCR of the two bacteria. The method was stable that 14 reference strains were tested and correctly identified and no cross interference. If the method wants to be applied in detecting food samples, the next step for searching and screening excellent enrichment solution to realize synchronous enrichment is needed.The rapid detection methods could accumulate scientific data for diagnosis, traceability and risk assessment of food-borne disease, provide the technical support for rapid detection of the two pathogens and rapid diagnosis of food poisoning,provide strong technical support dicision-making for the national food safety supervision department, make the foundation for bringing rapid detection methods into the national food safety standards... |
PDF Full Text Request